We used the bisulfite pyrosequencing method for methylation analyses of CASP8, FHIT, CDKN2B, and CHFR genes. Each primer was designed using Pyrosequencing Assay Design Software v2.0 (Qiagen, Germany). The primer sequence is listed in Supplementary Data 3 . The amplification was carried out according to the general guidelines suggested by pyrosequencing as follows: denaturing at 95°C for 10 min, followed by 45 cycles at 95°C for 30 s, at ∗ °C for 30 s, at 72°C for 30 s, and a final extension at 72°C for 5 min (∗CASP8 and FHIT: 54°C; CDKN2B and CHFR: 50°C). PCR products were confirmed by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining.
The ssDNA template was prepared from a biotinylated PCR product using streptavidin Sepharose® HP beads (Amersham Biosciences, Sweden) following the PSQ 96 sample preparation guide using multichannel pipettes. Fifteen picomoles of the respective sequencing primers were added for analysis. Sequencing was performed on a PyroMark ID system with the Pyro Gold reagent kit (Qiagen, Germany) according to the manufacturer's instructions. The methylation percentage was calculated as the average degree of methylation at CpG sites formulated by pyrosequencing.
The ssDNA template was prepared from a biotinylated PCR product using streptavidin Sepharose® HP beads (Amersham Biosciences, Sweden) following the PSQ 96 sample preparation guide using multichannel pipettes. Fifteen picomoles of the respective sequencing primers were added for analysis. Sequencing was performed on a PyroMark ID system with the Pyro Gold reagent kit (Qiagen, Germany) according to the manufacturer's instructions. The methylation percentage was calculated as the average degree of methylation at CpG sites formulated by pyrosequencing.